Abstract ? Mechanisms of pancreatic inflammation, tissue repair and carcinogenesis. The Pancreatic ductal adenocarcinoma, or pancreatic cancer, is one of the deadliest human malignancies, and little progress has been achieved in its treatment. Pancreatic cancer is characterized by an extensive fibrotic stroma, which includes vascular elements, infiltrating immune cells, extracellular matrix and a large number of fibroblasts responsible for creating a fibrotic microenvironment. The fibroblast population is presumed to derive from pancreatic stellate cells, although this notion has never been confirmed through lineage tracing studies due to the lack of appropriate tools. The role of the fibroblasts within the stroma is similarly controversial, with different studies ascribing a pro-tumor or anti- tumor function to these cells. Understanding the nature and biological role of pancreatic fibroblasts is necessary to set the stage for therapeutic strategies targeting the stroma as well as tumor cells. Although typically studied/treated as homogenous, the fibroblasts within the pancreatic stroma are a heterogeneous population, with potentially distinct functional properties. We have identified a sub-population of perivascular fibroblasts that are present within the normal pancreas and express the transcription factor Gli1, an effector and target gene of the Hedgehog signaling pathway. Moreover, we have identified a population of cells within the pancreas with mesenchymal stem cell (MSC) properties. Preliminary data indicate that these two populations might, completely or in part, overlap, and that they regulate the immune infiltration and polarization. The goal of this proposal is to functionally characterize the Gli1+ fibroblast population in pancreatic cancer. We will pursue this goal through 3 Specific Aims. In Aim 1, we will first lineage-trace perivascular Gli1+ fibroblasts in the normal pancreas to determine whether they contribute to the fibrotic reaction during carcinogenesis. Second, we will ablate this cell population at different stages of tumor development to determine whether they promote, or rather inhibit, tumor growth. In Aim 2, we will investigate the overlap between the MSC population and Gli1+ fibroblasts within the fibrotic pancreas, and study their ability to regulate immune function. Finally, in Aim 3, we will complement the other Aims by investigating the nature and role of Gli1 expressing immune cells. Throughout the Aims, we will determine whether the effects mediated by Gli1+ cells can be recapitulated by inhibition of Hedgehog signaling. These Aims will vertically enhance our understanding of the origin and function of the pancreatic cancer stroma, and might provide insight into the recent failure of clinical trials against the stroma, such as Hedgehog inhibition. The long-term goal of this application is to identify new cellular components that might be targeted therapeutically to benefit pancreatic cancer patients. Throughout the proposal, we will use a combination of in vitro and in vivo experiments, and include both genetically engineered mouse models of pancreatic cancer and human primary pancreatic cancer cells, with the goal of maximizing the translational potential of our findings. A large group of collaborators will be instrumental for the success of our work. The University of Michigan counts a large community of researchers dedicated to pancreatic cancer. Those include: experts in the clinical disease and a source of primary human samples (Dr Diane Simeone), an expert in both clinical and mouse modeling aspects of pancreatic cancer (Dr Andy Rhim), an expert in pancreas development, disease and cancer (Dr Howard Crawford). In addition, we collaborate with a leader in the field of cancer-associated mesenchymal stem cells (Drs Ron Buckanovich). The immunology component of this grant and in particular the bone marrow transplantation experiments will be performed with advice and support from Dr Ivan Maillard.In addition, the proposal will be supported by a number of outstanding Core facilities at the University of Michigan.